The overall goal of this research is to establish the role of intedeukin-8 in the genesis and persistence of endogenous pulmonary inflammation that occurs in this ovine model of smoke inhalation and bum injury. In Specific Aim 1, it will be determined if sequestered cells in the pulmonary circulation are initially activated in the bronchial circulation and contain F-actin. The bronchial circulation will be ablated and the pulmonary circulation examined for decreased sequestration of PMNs in pulmonary capillaries by confocal microscopy. Activation of polymorphonuclear leukocytes (PMNs) will be followed by flow cytometric measurement of blood and lung lymph PMN cell surface CD18 and L-selectin. PMN and red blood cell pulmonary transit times will be measured using both resting PMNs and PMNs from the injured animals. IL-8 and LTB4 will be determined in lung lymph and correlated with PARP activity. Specific Aim 2, usage of cell signaling pathways mediating activation of neutrophils and formation of F-actin in this injury are sought through measurement of total and phosphorylated JNK/SAPK, p38 Map kinase, ERK 1/2, Akt, and PARP activity by Western blotting, enzyme activity, and confocal laser scanning cytometry. Consequences of involvement of these pathways will be explored through translocation of NF-KappaB and AP-1 transcription factors in nuclear isolates of airway cells and neutrophils by electrophoretic mobility shift assays. IKappaB will be measured by Western blotting. These same molecules will be measured in individual cells in lung tissue by confocal microscopy and laser scanning cytometry to allow for identification of cell type and quantification of each molecule. In Specific Aim 3, the role of IL-8 in the genesis and maintenance of pulmonary inflammation secondary to smoke and bum injuries will be examined. Advantage will be taken of our construction of a combined CXCR1 and CXCR2 antagonist (AAR-IL-8) to directly test the role of IL-8 in pulmonary injury in smoke inhalation and bum trauma. AAR-IL-8 will be given intravenously as a constant infusion in injured sheep through 48 h. The activation status of neutrophils in lung lymph will be measured by flow cytometry. Laser scanning cytometry will be employed to characterize activation of neutrophils in lung tissue and to estimate obstructing neutrophils in pulmonary capillaries. Lung lymph flow, PaO2/FiO2, left atdal pressure, pulmonary artery and pulmonary, capillary wedge pressure, cardiac output, pulmonary microvascular and systemic vascular resistance will be measured.